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Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine
Vaccine xxx (2017) xxx–xxx
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Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine Fan Zhang, Maria Jun, Olivia Ledue, Muriel Herd, Richard Malley, Ying-Jie Lu ⇑ Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical School, Boston, MA, United States
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Article history: Received 20 December 2016 Received in revised form 29 May 2017 Accepted 30 May 2017 Available online xxxx Keywords: Staphylococcus aureus Vaccine Whole cell lysate
a b s t r a c t Staphylococcus aureus is a very important human pathogen that causes significant morbidity and mortality worldwide. Several vaccine clinical trials based on generating antibody against staphylococcal surface polysaccharides or proteins have been unsuccessful. A killed whole cell lysate preparation (SaWCA) was made by lysing a USA 300 strain with lysostaphin followed by sonication and harvest of the supernatant fraction. Immunization with SaWCA and cholera toxin (CT) generated robust IL-17A but relatively modest antibody responses, and provided protection in the skin abscess but not in the dermonecrosis or invasive infection model. In contrast, parenteral immunization with SaWCA and alum produced robust antibody and IL-17A responses and protected mice in all three models. Sera generated after immunization with SaWCA had measurable antibodies directed against six tested conserved surface proteins, and promoted opsonophagocytosis activity (OPA) against two S. aureus strains. Passive transfer of SaWCA-immune serum protected mice against dermonecrosis and invasive infection but provided no demonstrable effect against skin abscesses, suggesting that antibodies alone may not be sufficient for protection in this model. Thus, immunization with a SA lysate preparation generates potent antibody and T cell responses, and confers protection in systemic and cutaneous staphylococcal infection models. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction The Gram-positive bacterium Staphylococcus aureus is a common pathogen of humans that causes a wide range of infections, which can involve the skin (such as boils or cellulitis), as well as many other organs (including the lungs, heart, bone and joints, among others) or cause shock syndromes. The rise of methicillinresistance in S. aureus strains (MRSA), as well as the emergence of vancomycin-intermediate/resistant strains (VISA/VRSA) [1], increases the complexity and cost of treatment of these infections. It is estimated that, annually, 10 billion US dollars are spent treating hospital-associated infections (HAI), such as surgical site infections, central line associated bloodstream infections, ventilator associated pneumonia and catheter associated urinary tract infections. In the US, over 10% of HAI are likely due to infection by S. aureus [2,3]. While S. aureus can certainly cause disease in healthy individuals, those undergoing surgery, who are on dialysis, in intensive care units or with underlying immunocompromising conditions are at particularly high risk [4].
⇑ Corresponding author at: Division of Infectious Diseases, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, United States. E-mail address: [email protected] (Y.-J. Lu).
S. aureus vaccine development efforts have not been successful so far. While generating opsonophagocytic (OPA) antibody against the capsular polysaccharide of a microorganism has long been the vaccine strategy against pathogenic bacteria such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis, this approach has not been useful in the case of S. aureus: a candidate vaccine comprising two staphylococcal capsular polysaccharides (type 5 and 8) conjugated to recombinant exoprotein A showed partial protection in an early clinical trial but failed in a phase III clinical trial [5,6]. Similarly, a passive immunization trial using pooled human immunoglobulin preparations from donors with high antibody titers against staphylococcal CP 5 and 8 gave disappointing results [7]. Other vaccine approaches that have been tested but failed include active immunization with or passive transfer of either polyclonal or monoclonal antibodies to individual proteins of S. aureus [8–11]. Thus, there is a major and urgent unmet need for vaccine development against S. aureus. There is strong evidence to suggest that mechanisms other than antibodies alone may mediate resistance to staphylococcal infections. Indeed, whereas individuals with congenital agammaglobulinemia do not seem to be at particularly high risk for staphylococcal infections, children with complete DiGeorge syndrome (who lack a thymus and T cell responses) tend to have recurrent infections with S. aureus. Similarly, adult patients with HIV
http://dx.doi.org/10.1016/j.vaccine.2017.05.085 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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infection and CD4+ T cell deficiency are very susceptible to staphylococcal infection [12]. It was long known that patients with autosomal dominant hyper-IgE syndrome (Job’s syndrome) have a propensity to develop recurrent staphylococcal skin or pulmonary infections, but the underlying immunodeficiency has only been identified recently. It is now established that Job’s syndrome patients have mutations in STAT3, a signalling protein that is critical for the development of memory Th17 cells [13]. These data, while consistent with a role of T cells in immunity to staphylococcus, do not prove that CD4+ T cells, or Th17 cells in particular, play an independent role in preventing infection however, since CD4+ T cell deficiency or STAT3 mutations can also have important effects on antibody production or other aspects of the immune response. More recently, evidence supporting an independent role of IL-17A-mediated protective mechanisms against S. aureus infection has been obtained in mouse models. IL-17A-deficient mice are more prone to staphylococcal infection [14,15] and clearance of nasal staphylococcal carriage in mice is IL-17A dependent [16]. In support of this hypothesis, several protein-based vaccines (such as those containing IsdB or ClfA [17,18]) confer Th17dependent protection against S. aureus infection in preclinical models. Furthermore, adoptive transfer of immune Th17 cells can protect against infection [19,20], suggesting that these cells are sufficient to protect mice. More recently, studies in mice have also suggested a potential role of Th1 cells in protection [21,22]. Given the lack of success so far of immunization strategies relying exclusively on antibody generation and the growing evidence that T cells can provide protection against other extracellular respiratory pathogens [23–27], we hypothesize that an effective S. aureus vaccine strategy may require the generation of both antibody and T cell (specifically Th17 and Th1) responses to the organism. Killed staphylococcal whole cells have been tested as vaccine candidates in mice for protection against S. aureus infection but were not very successful [28,29]. A formalin-killed whole cell vaccine failed to protect animals in a rabbit endocarditis model. Whole cell vaccines made with irradiated wild-type and Spa mutant strains did not protect against subsequent intravenous infection with S. aureus. Recently, different whole cell preparations using mutant strains showed more promise in animal models. Immunization with an UV-irradiated preparation of a serine/threonine phosphatase mutant strain protected against systemic S. aureus infection [30]. Another study showed that previous immunization with a live strain containing a sortase A deletion protected against systemic infection [31]. In addition to intact whole cells, lysed cells have been shown to be protective in animal models [32–34]. It is important to note that these attempts were focused on antibody production against whole bacteria, and therefore T-cell (including Th17) responses, and their potential role in protection, were not evaluated. Here, we present evidence that a lysed S aureus whole cell vaccine preparation induces both antibody and Th17/Th1 responses when given with an adjuvant and can provide protection against S. aureus in three disease models. We show that while antiSaWCA antibody is sufficient for protection against skin dermonecrosis and invasive infection, passive transfer of antibodies did not protect against focal skin abscesses, arguing for an important and complimentary role of T-cells in protection following immunization with this vaccine.
2. Material and methods 2.1. Material Aluminum hydroxide (alum) was from Brenntag North America (2% Alhydrogel). Saline was from B. Braun Medical Inc. (Bethlehem,
PA). Cholera toxin (CT) was purchased from List Biological Laboratories. DMEM and FCS were from ThermoFisher scientific. Lysostaphin and other chemical reagents were purchased from Sigma. 2.2. Bacterial strains Staphylococcus aureus strains USA 300 TCH959 [35] and ATCC 29213 were purchased from ATCC. Bacteria were grown on blood agar plate overnight and then inoculated into Tryptic soy broth (TSB) to grow overnight at 37 °C with shaking. Cells were reinoculated into fresh TSB medium and incubated at 37 °C with shaking for 3 h. Cells were washed twice with saline and adjusted to concentrations as noted in animal models (described below) in saline before use. 2.3. SaWCA preparation The USA300 TCH959 strain was grown overnight on blood agar and resuspended into PBS. Cells were washed twice with PBS and resuspended to OD600 = 20 in PBS. Lysostaphin was added to the suspension and cells were shaken at 37 °C for 30 min. Cells were then lysed with sonication and then exposed to chloroform (1/40 vol/vol) and kept stirring at 4 °C for 2 h. Initially, we also included a preparation of cells that were only exposed to chloroform, without the initial lysing step. Either preparation was then plated on blood agar to confirm that no live bacteria was detectable before being frozen and then lyophilized. Lyophilized vials were reconstituted in the same volume of water and centrifuged for 5 min at 16,000g before use. For the lysed preparation, the supernatant fraction was isolated and defined as SaWCA. Protein concentrations were determined using the BCA kit with bovine serum albumin as standard (Thermo Scientific). 2.4. Immunization and challenge of mice All animal protocols were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital (protocol number 16-03-3133). Female C57BL/6 J mice (Jackson Laboratories, Bar Harbor, Maine) were used for all experiments. The age at time of first immunization was between 4 and 6 weeks. Two types of adjuvants were used in mice immunizations. Cholera toxin (CT) was used as an adjuvant in intranasal immunizations to promote T cell responses to SaWCA whereas aluminium hydroxide (alum) was used for subcutaneous (s.c.) immunizations to promote both antibody and T cell responses [36–39]. For intranasal immunization, SaWCA was mixed with CT and the dose of immunization was 64 lg (protein content) of SaWCA and 1 lg of CT per mouse per immunization. Mice were immunized twice one week apart and bled three weeks after last immunization. Peripheral blood samples were stimulated with SaWCA and assayed for IL-17A production; plasma samples were analyzed for antibody production by ELISA. For s.c. immunization, vaccines were prepared as follows. One day prior to immunization, SaWCA was diluted to the appropriate concentration, and mixed with alum at the indicated concentration; the mixture was then rotated end-over-end overnight at 4 °C to allow for adsorption. The immunization dose in s.c. experiment was 100 lg of SaWCA (protein content) per mouse. Gently restrained, nonanesthetized mice received 3 s.c. injections of 200 ml of adjuvant with or without antigen in the back at 2-week intervals. Blood was drawn 2 weeks after the last immunization; plasma and whole blood were assayed for antibody and for IL17A and INF-c production as noted above. Mice were challenged in three different models as described previously with some modifications [40–42]:
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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a. Skin abscess model. To generate abscesses, the back of mice was shaved one day prior to infection, then anesthetized and injected s.c. with 2–5 105 CFU of strain USA 300 (vaccine strain). Abscesses become readily visible and palpable under the skin within 1–2 days. Mice were sacrificed on day 4 and abscesses were transferred into 0.5 ml of PBS and homogenized. Bacteria were plated on Mannitol salt plates and colonies were counted after overnight culture at 37 °C. b. Dermonecrosis model. In this model, the same procedure was used as for the skin abscess model, but with a higher inoculum of 5 106 1 107 CFU of freshly grown strain USA 300 (vaccine strain). Mice formed lesion by day 2 or day 3 and were monitored for 14 days and the surface area of the skin lesions was determined using pictures processed via Image J software. c. Intravenous sepsis model. Mice were anesthetized and infected with 2.5 107 CFU of strain ATCC 29213 via retroorbital injection. Mice were monitored twice daily for illness for 14 days. Any ill-appearing animal (including any signs of ruffled fur, slow moving or closed eyes) was immediately and humanely euthanized. For histopathology staining, skin tissues were removed and fixed in 10% neutral buffered formalin, and then paraffinembedded. Sections were stained with hematoxylin and eosin at
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the pathology core facility of Harvard Medical School. Pictures were taken using an Olympus microscope. These three different models were used to test the efficacy of the vaccine. In the abscess model, the infection is restricted to a local area and usually circumscribed inside an abscess (without any evidence of dissemination to blood or organs) as shown in Fig. 1A and C, a situation which mimics one of the most common manifestations of staphylococcal infections in humans. In the dermonecrosis model, the mice have more destructive lesions (Fig. 1B and D). The invasive disease model uses the encapsulated strain (ATCC 29213), which has also been used to evaluate the potential candidate antigen Als3p [19]. Thus the use of the three models allows for testing vaccine efficacy test in models with differing pathophysiology, strains and inoculum size. 2.5. Antibody production and passive immunization studies New Zealand white rabbits were immunized three times with 1 mg of SaWCA and Freund’s adjuvant at Cocalico Biologicals (Reamstown, PA). We chose rabbit serum as a source of antibody in the passive transfer experiments because it has been traditionally used in mouse passive experiment studies involving S. aureus and also because of the volume of sera required [43,44]. IgG antibody level against SaWCA remain above 50% of input for up to two weeks (data not shown), which exceeds the duration of these
Fig. 1. Histology of skin abscess and dermonecrosis lesions. Mice were infected with either 2 105 CFU of USA 300 (A and C) or 1 107 CFU of USA 300 (B and D) subcutaneously. Mice were sacrificed on day 4 and histological slides prepared for examination of the abscess (A) and dermonecrosis sites (B) as shown by arrows. Mice skins were fixed in 10% formalin and Hemotoxylin and Eosin-stained for abscess (C) and dermonecrosis (D).
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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animal experiments. For passive transfer protection experiments, two different protocols were used. In the first, 500 ll of sera were given intraperitoneally 24 h before intravenous infection (sepsis model). To maximize any protective effect of antibodies, a second protocol was designed in which the bacterial inoculum was resuspended in 100 ll of sera for 30 min, after which the mixture of bacteria and sera was injected into mice (in all three models). 2.6. Enzyme-linked immunosorbent assay (ELISA) Assays for murine antibodies to whole bacteria were performed using Immulon 2 HB 96-microwell plates (Thermo Scientific, Waltham, MA) coated with SaWCA at 100 lg of protein per ml in PBS. Plates were blocked with 1% BSA in PBS. Antibody diluted in PBS with 0.05% Tween 20 (PBS-T) was added and incubated at room temperature for 2 h. Plates were washed with PBS-T, and secondary HRP-conjugated antibody to immunoglobulin G was added and incubated at room temperature for one hour. The plates were washed and developed with SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD). 2.7. Assay of IL-17A and IFN-c production in whole blood samples Fifty ml of heparinized blood were added to 450 ml DMEM (BioWhittaker, Walkersville, MD) containing 10% low-endotoxin defined FBS (Hyclone, Logan, UT), 50 mM 2-mercaptoethanol (Sigma) and ciprofloxacin (10 mg/ml, Cellgro, Manassas, VA). The cultures were incubated at 37 °C for 6 days with 10 lg/ml of SaWCA. Supernatants were collected following centrifugation and stored at -80 °C until analyzed by ELISA for IL-17A and INF-c (R&D Systems, Minneapolis, MN). For in vitro CD4+ and CD8+ depletion experiments, spleens and blood were obtained from immunized mice. Red blood cells were lysed with ACK lysis buffer (Thermo Fisher) and CD4+ or CD8+ cells were then depleted from the cell suspension with either anti-CD4+ or anti-CD8+ antibodylinked magnetic beads from Miltenyi Biotech following the manufacturer’s protocol. The remaining cells were plated at 1 106 cells/ml for PBMC and 1 107 cells/ml for splenocytes and stimulated with 10 lg/ml of SaWCA for 3 days before analysis for IL-17A production in the supernatant. 2.8. Opsonophagocytic killing assay (OPA) OPAs were performed as described previously [45] using differentiated HL-60 cells as a source of phagocytes. Briefly, USA 300 strain was grown as described above and resuspended to 2.5 104 CFU/ml in HBSS with 10% FBS. Eighty ll of bacteria (corresponding to about 2000 CFU) were first mixed with 10 ll of heatinactivated serum and incubated at room temperature for 30 min with shaking at 600 rpm. Ten ll of baby rabbit complement were added in the mixture. HL-60 cells that were differentiated using 0.8% N,N-dimethylformamide (DMF) were added at a cell:bacteria ratio of 100:1, shaken at 37 °C for another hour. Samples were diluted and plated on trypticase soy agar with 5% defibrinated sheep blood (TSA II) (BD) and incubated overnight at 37 °C with 5% CO2. Controls included bacteria treated in the absence of cells or with cells but no serum.
3. Results 3.1. Whole cell vaccine (SaWCA) preparations Killed non-lysed and lysed preparations were made as described above. In the case of the lysed preparation, the mixture was centrifuged at 16,000g for 5 min to remove intact cells and the supernatant fraction of killed whole cells was analyzed for protein composition on an SDS gel. The appearance of the supernatant fraction on the gel was virtually identical to that of the whole lysate (data not shown). Further analysis of the protein concentration by BCA showed that supernatant fraction retained about 80– 90% of the total protein amount (data not shown). 3.2. Lysed SaWCA is immunogenic following immunization by either the intranasal or subcutaneous route Our experience with S. pneumoniae suggested that intranasal immunization with killed cells adjuvanted with CT (an adjuvant known to promote Th17 responses [39,46]) could generate robust Th17 responses to protein antigens [47]. Therefore we began our experiments by testing whether the same would be observed with a killed staphylococcal whole cell vaccine. Surprisingly, as shown in the supplemental figure, although mice immunized with the killed non-lysed preparation intranasally twice a one-week interval generated higher Th17 response than CT-immunized mice, the level of IL-17A elicited by peripheral blood was quite low (median 25 pg/ml, see supplementary figure). In contrast, blood from mice that received the lysed preparation of SaWCA with CT expressed much higher IL-17A (median value 1640 pg/ml), also significantly greater than that of mice that received CT alone (median 8 pg/ml, P < 0.0001) (Fig. 2A). Given the reported importance of IL-17A in protection against staphylococcal infection [16,20], we selected the lysed preparation for all subsequent studies (which will be referred to as SaWCA). INF-c production by the blood of mice from the SaWCA/CT group after stimulation was significantly higher than that of mice that received CT alone, but overall low (median 22 pg/ml median 9.77 pg/ml, P = 0.015). The sera of SaWCA/CT-immunized mice had about 10 times higher anti-SaWCA antibody levels compared to the sera from mice immunized with CT (P < 0.001, Fig. 2B). Because we wished to elicit both robust antibody and T cell responses, subcutaneous immunization with alum as adjuvant was also evaluated, as we have previously shown that the addition of alum to pneumococcal whole cells enhanced cellular immune responses while also eliciting high antibody levels [48]. Mice subcutaneously immunized with SaWCA/alum generated significantly higher IL-17A (median 625 pg/ml) and IFN-c (median 48 pg/ml) responses than control mice (median 8 pg/ml and 8.54 pg/ml, P < 0.0001 and P < 0.05 respectively) (Fig. 2C). The antibody response to SaWCA was also significantly greater (by over 1000fold) in the SaWCA/alum-immunized compared to the control group, and 100 times higher than that observed following intranasal immunization with SaWCA and CT (Fig. 2D). The differential antibody response following intranasal vs. parenteral immunization with preserved T cell responses in both groups also provided an opportunity to evaluate the role of vaccine-elicited antibodies in each of the models.
2.9. Statistical analysis 3.3. IL-17A is generated by CD4+ cells Serum antibody concentrations, IL-17A and IFN-c release from peripheral cells and CFU counts in abscesses were compared between groups using the Mann-Whitney U test using PRISM (version 6.0 g, GraphPad Software, Inc). Differences in survival were analyzed by the Mantel-Cox test, using PRISM as well.
CD4+, CD8+, and cd T cells as well as innate lymphoid cells have all been shown to produce IL-17A [49–51]. We thus analyzed which cell type is the main source for IL-17A production in SaWCA-immunized mice. Blood PBMC or splenocytes from immu-
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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production whereas depletion of CD8+ cells did not have any effect on IL-17A production. This result clearly suggests that IL-17A was mainly made from CD4+ T cells.
3.4. Binding and opsonophagocytic activity of sera obtained from mice immunized with SaWCA We then analyzed whether serum from SaWCA/alumimmunized mice generated antibody against staphylococcal antigens. We chose to evaluate the responses to 6 conserved antigens/virulence factors that had been previously shown to contribute to protection in various animal models: ClfA: clumping factor A; ClfB: clumping factor B; SdrD: SD-repeating containing protein D; a-toxin: alpha hemolysin; IsdA: Iron-regulated surface determinant protein A; IsdB: Iron-regulated surface determinant protein B [9,52,53]. As shown in Fig. 3A, SaWCA/alum immune serum demonstrated robust antibody responses to 3 proteins: SdrD, ClfA and ClfB and weaker, but still detectable, antibody responses to the other 3 proteins. Next we evaluated whether the sera from mice immunized with SaWCA/alum demonstrate in vitro killing activity. Two bacterial strains (USA 300 (vaccine strain) and ATCC 29213 (heterogenic strain)) were incubated first with heat-inactivated serum from
Fig. 2. Immune responses following intranasal and subcutaneous immunization with SaWCA. Blood samples from immunized mice were stimulated with 10 lg/ml (protein content) SaWCA for six days. IL-17A and INF-c were measured from cell supernatants for intranasal immunization (A) and subcutaneous immunization (C). Serum antibody against SaWCA was determined by ELISA following intranasal immunization (B) or subcutaneous immunization (D). E. Splenocytes or PBMC were depleted of CD4+ or CD8+ cells and stimulated with SaWCA for 3 days. Open bars represent cells without depletion, grey bars represent responses from cells lacking CD4+ cells and black bars represent responses from cells lacking CD8+ cells. Statistical analysis was done using the Mann-Whitney U test: *** P < 0.001, **** P < 0.0001.
nized mice were depleted with either CD4+ or CD8+ cells, and then stimulated with SaWCA. As shown in Fig. 2E, depletion of CD4+ cells totally abolished antigen-dependent stimulation of IL-17A
Fig. 3. Serological and functional analysis of immune responses following immunization with SaWCA and alum. (A) Antibody fold-rise in immunized mice against 6 selected surface proteins were determined by ELISA. (B) Opsonophagocytic killing assay using serum from mice immunized with SaWCA and alum vs. alum alone. Percentage of surviving bacteria from each sample is presented. Statistical analysis was performed using the Mann-Whitney U test: *** P < 0.001, **** P < 0.0001.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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are generated that not only can recognize important virulence factors of S. aureus, but also promote killing of S. aureus by neutrophils. 3.5. Protection by immunization with SaWCA in the three infection models
Fig. 4. Protection against skin abscess using SaWCA administered intranasally (A) or parenterally (B). The back of mice was shaved and mice subsequently challenged with 2 105 CFU of USA 300 for 4 days. Bacterial CFUs in abscesses were determined by serial dilutions. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Statistical analysis was done by MannWhitney U test: * P < 0.05, ** P < 0.01.
mice immunized either with alum or SaWCA/alum and then incubated with complement and HL-60-derived neutrophils. As shown in Fig. 3B, sera from mice immunized with SaWCA/alum killed 30% more bacteria than the control serum for both strains (P < 0.0001). Thus, following immunization with SaWCA and alum, antibodies
We first tested protection by SaWCA in the mouse abscess model. Immunization of mice with SaWCA/CT significantly reduced the bacterial density recovered from the abscess 4 days after infection compared to exposure to CT alone (Fig. 4A). Similarly, mice in the SaWCA/alum group also had a lower incidence of abscess compared to mice that received alum alone (10% vs. 70%, P = 0.02) and significantly fewer staphylococcal colonies could be recovered from the lesions when these were present (geometric mean 62 vs. 2.5 104 cfu, P = 0.0079) (Fig. 4B). In the dermonecrosis model, no protection was observed in mice immunized with SaWCA/CT compared to mice that received CT alone (Fig. 5A). In contrast, SaWCA/alum was clearly protective: the peak sizes (day 7) of the necrotic lesions were significantly lower in the SaWCA/alum-immunized mice compared to the controls (0.02 vs. 0.372 cm2, P = 0.0067), and similar differences were notable at most other measured time points (Fig. 5B). We have also examined skin pathology in the alum and SaWCA/alum immunized mice four days post infection. As shown in Fig. 5C, mice exposed to alum alone had disrupted skin tissue at the infection site and little infiltration of neutrophils can be observed. In contrast, SaWCA/ alum-immunized mice had intact skin tissue but an abscess that contains neutrophils and bacteria can be readily seen (Fig. 5D). We then evaluated whether protection against invasive disease following retroorbital injection of S. aureus would differ based on the immunization route. No protection was observed following
Fig. 5. Protection against dermonecrosis following immunization with SaWCA administered intranasally (A) or parenterally (B). Mice were infected with 1 107 CFU of USA 300 and the area of skin dermonecrosis was measured at different time points as indicated. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Skin histopathology with H&E staining was also performed on mice that received either alum (C) or SaWCA/alum (D) at four days post infection. Statistical analysis was calculated by Mann-Whitney U. * P < 0.05, ** P < 0.01, *** P < 0.001.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
F. Zhang et al. / Vaccine xxx (2017) xxx–xxx
intranasal immunization with SaWCA/CT (Fig. 6A) whereas SaWCA/alum-immunized mice had a significantly higher survival rate than mice immunized with alum alone (Fig. 6B, p = 0.045). Taken together, the result of these experiments suggest that antibodies, which are more robustly generated following parenteral immunization, play an important role in the dermonecrosis and sepsis models, but may be less critical in the abscess model. To evaluate this possibility further, we proceeded to evaluate whether protection could be achieved by passive immunization using sera directed against the lysate preparation.
3.6. Protection by passive transfer of immune serum To evaluate the role of antibodies in the different models used, we began our studies by injecting mice one day before infection with 500 ll of sera obtained from rabbits either before or after three immunizations with SaWCA. Although there was a trend towards delayed death (P = 0.07 by Kaplan-Meier) no difference in overall survival between the two groups of mice could be detected (2/10 vs. 0/10 survivors, Fig. 7A). To try to maximize protection provided by passive transfer, we pre-mixed bacteria with either pre- or post-immunization serum before infection. With this form of passive transfer, as shown in Fig. 7B, there was significant protection by immune serum (p = 0.03). The protective effect of immune serum clearly diminishes after 4 days, potentially due to the limited amount of antibody provided via this route (100 ll).
Fig. 6. Protection against sepsis following immunization with SaWCA administered intranasally (A) or parenterally (B). Mice were infected with 2.5 107 CFU ATCC 29213 intravenously and sickness was monitored for 14 days. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Statistical analysis of the survival curves was performed by the Mantel-Cox test: * P < 0.05.
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Next we evaluated passive transfer of immune serum in the two other models. Immune serum did not protect mice in the skin abscess model with either infusion of 500 ll of immune serum one day prior to infection or bacteria pre-mixed with 100 ll of immune serum (Fig. 7C). When mice were challenged in the dermonecrosis model, mice that received immune serum had significantly better protection than that received pre-immunization serum after day 5, even though these mice had similar size of lesions early on (day 3 and 4) (Fig. 7D). Thus we conclude that antibodies directed against SaWCA contribute to protection in the sepsis and dermonecrosis model, whereas no effect of antibodies could be detected in the abscess model. 4. Discussion Given the morbidity and mortality associated with staphylococcal infections and the rise in antimicrobial resistance, there is an important need for the development of a vaccine for S. aureus. There is a growing consensus that a vaccine containing multiple components is in all likelihood required to combat this pathogen, given the complexity of the various toxins and other virulence factors produced by S. aureus [9,52–55]. Here, we present data using a lysed whole cell vaccine that is able to generate staphylococcusspecific antibody, Th17 and Th1 responses, and provide protection in three different mouse challenge models including a bacterial strain unrelated to the vaccine strain. Antibodies to key virulence factors were detectable in serum following immunization; the serum also promoted complement-mediated killing to two staphylococcal strains and was sufficient to confer protection against dermonecrosis and invasive disease. Whole cell vaccine preparations against S. aureus have been evaluated for protection in various preclinical models with marginal success, with more promising results when mutant strains (Spa or sortase mutants) were used [29–32]. A heat-killed whole cell vaccine was able to protect against intraperitoneal infection by live S. aureus and promoted phagocytosis [56]. A whole cell lysate vaccine containing 5 different strains of S. aureus designed to reduce dairy cattle mastitis has been commercially available for veterinary use in Europe [57,58]. Our strategy for whole cell preparation was also to lyse the bacteria to enhance immunogenicity, but in our case not by phage but by sonication. A key difference between our strategy and those employed previously is the introduction of adjuvants to promote both Th17 and Th1 responses. In the course of our work on the development of a whole cell or defined antigen pneumococcal vaccine, we found that when given with either alum hydroxide or CT, antigens elicited both antibody and Th17/Th1 responses, resulting in antibody-mediated protection against sepsis and IL-17A-mediated protection against nasopharyngeal colonization by S. pneumoniae [37,48,59,60]. Similarly, here, the inclusion of alum via the parenteral route induced Tcell and antibody responses whereas intranasal immunization with SaWCA and CT induced robust T-cell but only modest antibody responses. These results are consistent with those from our own group and that of others, in which intranasal immunization with CT elicits production of IL-17A and IFN- c but is less effective at generating systemic antibody responses than systemic immunization [36–39]. Alum-based adjuvants have being safely used in human for decades and avoid the potential safety issue of using intranasal cholera toxin mutants intranasally. Protection by SaWCA was tested against the original vaccine strain (USA 300) in both the abscess and dermonecrosis models and also against a heterogenic strain (29213), suggesting that protection is not restricted to the vaccine strain. However, protection against additional staphylococcal isolates needs to be tested to examine whether the protection by SaWCA is as broad as in the case of the pneumococcal whole cell vaccine [61].
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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Fig. 7. Protection by passive transfer of sera. Mice were passively immunized with either pre-immune serum (closed squares) or post-immune serum (open squares) and then challenged in the sepsis model (A). Another two groups of mice were infected with strain 29213 resuspended in either pre-immune serum or post-immune serum (B). Similarly, mice were passively immunized with either pre-serum (closed squares) or post-immune serum (open squares) and then challenged in the abscess model (C), dermonecrosis model (D). Comparison of survival curves was performed using the Kaplan-Meier test; differences in bacterial CFUs and size of dermonecrotic lesions were analyzed using the Mann-Whitney U test.
Interestingly, the comparison of the two types of immunization suggests a differential role of antibodies and T cell responses in the two skin models we employed. Whereas both types of immunization resulted in protection against skin abscess, intranasal immunization with CT (which elicits comparable T cell responses, but significantly lower antibody responses than parenteral immunization with alum) did not provide significant protection against dermonecrosis, suggesting that antibodies may be most important in preventing this type of pathology. Along these lines, transfer of immune serum conferred protection in the dermonecrosis and invasive disease models, consistent with an important role of humoral immunity in preventing these infections. Conversely, the fact that passive transfer of immune serum did not protect mice in the abscess model supports the hypothesis that antibody is not critical in controlling this type of infection and that T-cell immunity may be the major factor for protection against skin abscesses. Another interesting implication of this study is that it may be possible to use the SaWCA as a probe for the identification of protective staphylococcal antigens, as we have done previously with S. pneumoniae [25,62]. The whole cell vaccine likely contains over 2000 staphylococcal proteins; thus, while quantification of each antigen in the whole cell vaccine and determination of their role in protection would represent a major challenge, more focused studies can be performed to examine a putative role of individual antigens of interest. For example, we were able to detect antibody
responses to a number of previously identified putative protective antigens: ClfA/B (fibrinogen-binding proteins that mediate attachment of S. aureus to host cells [63,64]), SdrD (a surface-associated calcium-binding protein against which antibodies have OPA) and Hla (alpha-hemolysin, an important virulence factor for skin dermonecrosis). Examination of preparations lacking these antigens could provide important information on their potential role in protection. We did not detect robust antibody responses against IsdA and IsdB, which suggests that an antibody response to these proteins may not be strictly necessary for protection against skin disease. Overall, we noted that while there was an over 1000 fold rise in antistaphylococcal antibody titer, the increase in killing capacity of the serum was only 30%, suggesting that only some of these antibodies contribute to OPA and that there are other antibodymediated mechanisms that provide protection. At the same time, it is conceivable that optimal protection against this organism may require eliciting both antibodies and T cell responses to a large number of antigens, which would be difficult to achieve with purified antigenic components. While killed whole cell vaccines are often considered an antiquated immunization approach, recent experience with S. pneumoniae suggests that the preclinical and clinical development of such a vaccine is feasible and promising [25,62,65]. Whole cell constructs also have the advantage of providing auto-adjuvanting effects which may enhance their immunogenicity and potency [59]. Thus, our
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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approach may have immunological advantages that should be further examined. Funding information This work was Pharmaceuticals.
supported
by
a
grant
from
Takeda
Acknowledgements YJL gratefully acknowledges support from Boston Children’s Hospital faculty development award and NIH grant R21AI103480 from the National Institute of Allergy and Infectious Diseases. RM gratefully acknowledges support from the Translational Research Program at Boston Children’s Hospital and NIH grant R01 AI100114. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.05. 085. References [1] Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J Clin Invest 2014;124:2836–40. [2] Zimlichman E, Henderson D, Tamir O, Franz C, Song P, Yamin CK, et al. Health care-associated infections: a meta-analysis of costs and financial impact on the US health care system. JAMA Int Med 2013;173:2039–46. [3] Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA, et al. Multistate point-prevalence survey of health care-associated infections. N Engl J Med 2014;370:1198–208. [4] Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Inf Dis 2005;5:751–62. [5] Shinefield H, Black S, Fattom A, Horwith G, Rasgon S, Ordonez J, et al. Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis. N Engl J Med. 2002;346:491–6. [6] Fattom A, Matalon A, Buerkert J, Taylor K, Damaso S, Boutriau D. Efficacy profile of a bivalent Staphylococcus aureus glycoconjugated vaccine in adults on hemodialysis: Phase III randomized study. Human vaccines & immunotherapeutics. 2015;11:632–41. [7] Rupp ME, Holley Jr HP, Lutz J, Dicpinigaitis PV, Woods CW, Levine DP, et al. Phase II, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-Staphylococcus aureus capsular polysaccharide immune globulin in treatment of Staphylococcus aureus bacteremia. Antimicrob Agents Chemother 2007;51:4249–54. [8] Fowler VG, Allen KB, Moreira ED, Moustafa M, Isgro F, Boucher HW, et al. Effect of an investigational vaccine for preventing Staphylococcus aureus infections after cardiothoracic surgery: a randomized trial. JAMA 2013;309:1368–78. [9] Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: Staphyloccocus aureus vaccines: problems and prospects. Vaccine. 2013;31:2723–30. [10] DeJonge M, Burchfield D, Bloom B, Duenas M, Walker W, Polak M, et al. Clinical trial of safety and efficacy of INH-A21 for the prevention of nosocomial staphylococcal bloodstream infection in premature infants. J Pediatr. 2007;151:260–5. 5 e1. [11] Weisman LE, Thackray HM, Steinhorn RH, Walsh WF, Lassiter HA, Dhanireddy R, et al. A randomized study of a monoclonal antibody (pagibaximab) to prevent staphylococcal sepsis. Pediatrics 2011;128:271–9. [12] Crum-Cianflone N, Weekes J, Bavaro M. Recurrent community-associated methicillin-resistant Staphylococcus aureus infections among HIV-infected persons: incidence and risk factors. AIDS Patient Care STDs 2009;23:499–502. [13] Ma CS, Chew GY, Simpson N, Priyadarshi A, Wong M, Grimbacher B, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J Exp Med 2008;205:1551–7. [14] Ishigame H, Kakuta S, Nagai T, Kadoki M, Nambu A, Komiyama Y, et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009;30:108–19. [15] Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, et al. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest 2010;120:1762–73. [16] Archer NK, Harro JM, Shirtliff ME. Clearance of Staphylococcus aureus nasal carriage is T cell dependent and mediated through interleukin-17A expression and neutrophil influx. Infect Immun 2013;81:2070–5.
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[17] Joshi A, Pancari G, Cope L, Bowman EP, Cua D, Proctor RA, et al. Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model. Human Vaccines Immunother 2012;8:336–46. [18] Narita K, Hu DL, Mori F, Wakabayashi K, Iwakura Y, Nakane A. Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A. Infect Immun 2010;78:4234–42. [19] Spellberg B, Ibrahim AS, Yeaman MR, Lin L, Fu Y, Avanesian V, et al. The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infect Immun 2008;76:4574–80. [20] Montgomery CP, Daniels M, Zhao F, Alegre ML, Chong AS, Daum RS. Protective immunity against recurrent Staphylococcus aureus skin infection requires antibody and interleukin-17A. Infect Immun 2014;82:2125–34. [21] Choi SJ, Kim MH, Jeon J, Kim OY, Choi Y, Seo J, et al. Active immunization with extracellular vesicles derived from Staphylococcus aureus effectively protects against staphylococcal lung infections, mainly via Th1 cell-mediated immunity. PLoS One 2015;10:e0136021. [22] Brown AF, Murphy AG, Lalor SJ, Leech JM, O’Keeffe KM, Mac Aogain M, et al. Memory Th1 cells are protective in invasive Staphylococcus aureus infection. PLoS Pathog 2015;11:e1005226. [23] Glennie ND, Yeramilli VA, Beiting DP, Volk SW, Weaver CT, Scott P. Skinresident memory CD4+ T cells enhance protection against Leishmania major infection. J Exp Med 2015;212:1405–14. [24] Zhang Z, Clarke TB, Weiser JN. Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. J Clin Invest 2009;119:1899–909. [25] Moffitt KL, Gierahn TM, Lu YJ, Gouveia P, Alderson M, Flechtner JB, et al. T(H) 17-based vaccine design for prevention of Streptococcus pneumoniae colonization. Cell Host Microbe 2011;9:158–65. [26] Malley R. Antibody and cell-mediated immunity to Streptococcus pneumoniae: implications for vaccine development. J Mol Med 2010;88:135–42. [27] Lu YJ, Gross J, Bogaert D, Finn A, Bagrade L, Zhang Q, et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog 2008;4:e1000159. [28] Greenberg DP, Ward JI, Bayer AS. Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits. Infect Immun 1987;55:3030–4. [29] van Diemen PM, Yamaguchi Y, Paterson GK, Rollier CS, Hill AV, Wyllie DH. Irradiated wild-type and Spa mutant Staphylococcus aureus induce anti-S. aureus immune responses in mice which do not protect against subsequent intravenous challenge. Pathogens Dis 2013;68:20–6. [30] Burnside K, Lembo A, Harrell MI, Klein JA, Lopez-Guisa J, Siegesmund AM, et al. Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection. J Infect Dis 2012;206:1734–44. [31] Kim HK, Kim HY, Schneewind O, Missiakas D. Identifying protective antigens of Staphylococcus aureus, a pathogen that suppresses host immune responses. FASEB J: Off Pub Fed Am Soc Exp Biol 2011;25:3605–12. [32] Camussone CM, Veaute CM, Pujato N, Morein B, Marcipar IS, Calvinho LF. Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant. Res Vet Sci 2014;96:86–94. [33] O’Brien CN, Guidry AJ, Douglass LW, Westhoff DC. Immunization with Staphylococcus aureus lysate incorporated into microspheres. J Dairy Sci 2001;84:1791–9. [34] Giese MJ, Adamu SA, Pitchekian-Halabi H, Ravindranath RM, Mondino BJ. The effect of Staphylococcus aureus phage lysate vaccine on a rabbit model of staphylococcal blepharitis, phlyctenulosis, and catarrhal infiltrates. Am J Ophthalmol 1996;122:245–54. [35] Highlander SK, Hulten KG, Qin X, Jiang H, Yerrapragada S, Mason Jr EO, et al. Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus. BMC Microbiol 2007;7:99. [36] Lu YJ, Zhang F, Sayeed S, Thompson CM, Szu S, Anderson PW, et al. A bivalent vaccine to protect against Streptococcus pneumoniae and Salmonella typhi. Vaccine 2012;30:3405–12. [37] Lu YJ, Yadav P, Clements JD, Forte S, Srivastava A, Thompson CM, et al. Options for inactivation, adjuvant, and route of topical administration of a killed, unencapsulated pneumococcal whole-cell vaccine. Clin Vaccine Immunol 2010;17:1005–12. [38] Lee JB, Jang JE, Song MK, Chang J. Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens. PLoS One 2009;4: e5190. [39] Mattsson J, Schon K, Ekman L, Fahlen-Yrlid L, Yrlid U, Lycke NY. Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsalpha in CD11b(+) DCs. Mucosal Immunol 2015;8:815–27. [40] Kim HK, Missiakas D, Schneewind O. Mouse models for infectious diseases caused by Staphylococcus aureus. J Immunol Methods 2014;410:88–99. [41] Molne L, Tarkowski A. An experimental model of cutaneous infection induced by superantigen-producing Staphylococcus aureus. J Invest Dermatol 2000;114:1120–5. [42] Krut O, Utermohlen O, Schlossherr X, Kronke M. Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates. Infect Immun 2003;71:2716–23.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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F. Zhang et al. / Vaccine xxx (2017) xxx–xxx
[43] Mishra RP, Mariotti P, Fiaschi L, Nosari S, Maccari S, Liberatori S, et al. Staphylococcus aureus FhuD2 is involved in the early phase of staphylococcal dissemination and generates protective immunity in mice. J Infect Dis 2012;206:1041–9. [44] McAdow M, Kim HK, Dedent AC, Hendrickx AP, Schneewind O, Missiakas DM. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood. PLoS Pathog 2011;7:e1002307. [45] Bagnoli F, Fontana MR, Soldaini E, Mishra RP, Fiaschi L, Cartocci E, et al. Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus. Proc Natl Acad Sci USA 2015;112:3680–5. [46] Larena M, Holmgren J, Lebens M, Terrinoni M, Lundgren A. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling. J Immunol 2015;194:3829–39. [47] Malley R, Lipsitch M, Stack A, Saladino R, Fleisher G, Pelton S, et al. Intranasal immunization with killed unencapsulated whole cells prevents colonization and invasive disease by capsulated pneumococci. Infect Immun 2001;69:4870–3. [48] Lu YJ, Leite L, Goncalves VM, Dias Wde O, Liberman C, Fratelli F, et al. GMPgrade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis. Vaccine 2010;28:7468–75. [49] Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004;21:467–76. [50] Xu H, Wang X, Liu DX, Moroney-Rasmussen T, Lackner AA, Veazey RS. IL-17producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques. Mucosal Immunol 2012;5:658–69. [51] Gladiator A, Wangler N, Trautwein-Weidner K, LeibundGut-Landmann S. Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection. J Immunol 2013;190:521–5. [52] Spellberg B, Daum R. Development of a vaccine against Staphylococcus aureus. Semin Immunopathol 2012;34:335–48. [53] Proctor RA. Challenges for a universal Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:1179–86. [54] Proctor RA. Is there a future for a Staphylococcus aureus vaccine? Vaccine 2012;30:2921–7.
[55] Daum RS, Spellberg B. Progress toward a Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:560–7. [56] Koenig MG, Melly MA, Rogers DE. Factors relating to the virulence of Staphylococci. III. Antibacterial versus antioxic immunity. J Exp Med 1962;116:601–10. [57] Luby CD, Middleton JR, Ma J, Rinehart CL, Bucklin S, Kohler C, et al. Characterization of the antibody isotype response in serum and milk of heifers vaccinated with a Staphylococcus aureus bacterin (Lysigin). J Dairy Res 2007;74:239–46. [58] Middleton JR, Ma J, Rinehart CL, Taylor VN, Luby CD, Steevens BJ. Efficacy of different Lysigin formulations in the prevention of Staphylococcus aureus intramammary infection in dairy heifers. J Dairy Res 2006;73:10–9. [59] Moffitt K, Skoberne M, Howard A, Gavrilescu LC, Gierahn T, Munzer S, et al. Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins. Infect Immun 2014;82:2079–86. [60] Moffitt KL, Yadav P, Weinberger DM, Anderson PW, Malley R. Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine. Vaccine 2012;30:4316–22. [61] Malley R, Anderson PW. Serotype-independent pneumococcal experimental vaccines that induce cellular as well as humoral immunity. Proc Natl Acad Sci USA 2012;109:3623–7. [62] Moffitt KL, Malley R, Lu YJ. Identification of protective pneumococcal T(H)17 antigens from the soluble fraction of a killed whole cell vaccine. PLoS One 2012;7:e43445. [63] O’Brien L, Kerrigan SW, Kaw G, Hogan M, Penades J, Litt D, et al. Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB, the serine-aspartate repeat protein SdrE and protein A. Mol Microbiol 2002;44:1033–44. [64] McDevitt D, Francois P, Vaudaux P, Foster TJ. Identification of the ligandbinding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol 1995;16:895–907. [65] Moffitt KL, Malley R. Next generation pneumococcal vaccines. Curr Opin Immunol 2011;23:407–13.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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Vaccine journal homepage: www.elsevier.com/locate/vaccine
Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine Fan Zhang, Maria Jun, Olivia Ledue, Muriel Herd, Richard Malley, Ying-Jie Lu ⇑ Division of Infectious Diseases, Boston Children’s Hospital, Harvard Medical School, Boston, MA, United States
a r t i c l e
i n f o
Article history: Received 20 December 2016 Received in revised form 29 May 2017 Accepted 30 May 2017 Available online xxxx Keywords: Staphylococcus aureus Vaccine Whole cell lysate
a b s t r a c t Staphylococcus aureus is a very important human pathogen that causes significant morbidity and mortality worldwide. Several vaccine clinical trials based on generating antibody against staphylococcal surface polysaccharides or proteins have been unsuccessful. A killed whole cell lysate preparation (SaWCA) was made by lysing a USA 300 strain with lysostaphin followed by sonication and harvest of the supernatant fraction. Immunization with SaWCA and cholera toxin (CT) generated robust IL-17A but relatively modest antibody responses, and provided protection in the skin abscess but not in the dermonecrosis or invasive infection model. In contrast, parenteral immunization with SaWCA and alum produced robust antibody and IL-17A responses and protected mice in all three models. Sera generated after immunization with SaWCA had measurable antibodies directed against six tested conserved surface proteins, and promoted opsonophagocytosis activity (OPA) against two S. aureus strains. Passive transfer of SaWCA-immune serum protected mice against dermonecrosis and invasive infection but provided no demonstrable effect against skin abscesses, suggesting that antibodies alone may not be sufficient for protection in this model. Thus, immunization with a SA lysate preparation generates potent antibody and T cell responses, and confers protection in systemic and cutaneous staphylococcal infection models. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction The Gram-positive bacterium Staphylococcus aureus is a common pathogen of humans that causes a wide range of infections, which can involve the skin (such as boils or cellulitis), as well as many other organs (including the lungs, heart, bone and joints, among others) or cause shock syndromes. The rise of methicillinresistance in S. aureus strains (MRSA), as well as the emergence of vancomycin-intermediate/resistant strains (VISA/VRSA) [1], increases the complexity and cost of treatment of these infections. It is estimated that, annually, 10 billion US dollars are spent treating hospital-associated infections (HAI), such as surgical site infections, central line associated bloodstream infections, ventilator associated pneumonia and catheter associated urinary tract infections. In the US, over 10% of HAI are likely due to infection by S. aureus [2,3]. While S. aureus can certainly cause disease in healthy individuals, those undergoing surgery, who are on dialysis, in intensive care units or with underlying immunocompromising conditions are at particularly high risk [4].
⇑ Corresponding author at: Division of Infectious Diseases, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, United States. E-mail address: [email protected] (Y.-J. Lu).
S. aureus vaccine development efforts have not been successful so far. While generating opsonophagocytic (OPA) antibody against the capsular polysaccharide of a microorganism has long been the vaccine strategy against pathogenic bacteria such as Haemophilus influenzae type b, Streptococcus pneumoniae and Neisseria meningitidis, this approach has not been useful in the case of S. aureus: a candidate vaccine comprising two staphylococcal capsular polysaccharides (type 5 and 8) conjugated to recombinant exoprotein A showed partial protection in an early clinical trial but failed in a phase III clinical trial [5,6]. Similarly, a passive immunization trial using pooled human immunoglobulin preparations from donors with high antibody titers against staphylococcal CP 5 and 8 gave disappointing results [7]. Other vaccine approaches that have been tested but failed include active immunization with or passive transfer of either polyclonal or monoclonal antibodies to individual proteins of S. aureus [8–11]. Thus, there is a major and urgent unmet need for vaccine development against S. aureus. There is strong evidence to suggest that mechanisms other than antibodies alone may mediate resistance to staphylococcal infections. Indeed, whereas individuals with congenital agammaglobulinemia do not seem to be at particularly high risk for staphylococcal infections, children with complete DiGeorge syndrome (who lack a thymus and T cell responses) tend to have recurrent infections with S. aureus. Similarly, adult patients with HIV
http://dx.doi.org/10.1016/j.vaccine.2017.05.085 0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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infection and CD4+ T cell deficiency are very susceptible to staphylococcal infection [12]. It was long known that patients with autosomal dominant hyper-IgE syndrome (Job’s syndrome) have a propensity to develop recurrent staphylococcal skin or pulmonary infections, but the underlying immunodeficiency has only been identified recently. It is now established that Job’s syndrome patients have mutations in STAT3, a signalling protein that is critical for the development of memory Th17 cells [13]. These data, while consistent with a role of T cells in immunity to staphylococcus, do not prove that CD4+ T cells, or Th17 cells in particular, play an independent role in preventing infection however, since CD4+ T cell deficiency or STAT3 mutations can also have important effects on antibody production or other aspects of the immune response. More recently, evidence supporting an independent role of IL-17A-mediated protective mechanisms against S. aureus infection has been obtained in mouse models. IL-17A-deficient mice are more prone to staphylococcal infection [14,15] and clearance of nasal staphylococcal carriage in mice is IL-17A dependent [16]. In support of this hypothesis, several protein-based vaccines (such as those containing IsdB or ClfA [17,18]) confer Th17dependent protection against S. aureus infection in preclinical models. Furthermore, adoptive transfer of immune Th17 cells can protect against infection [19,20], suggesting that these cells are sufficient to protect mice. More recently, studies in mice have also suggested a potential role of Th1 cells in protection [21,22]. Given the lack of success so far of immunization strategies relying exclusively on antibody generation and the growing evidence that T cells can provide protection against other extracellular respiratory pathogens [23–27], we hypothesize that an effective S. aureus vaccine strategy may require the generation of both antibody and T cell (specifically Th17 and Th1) responses to the organism. Killed staphylococcal whole cells have been tested as vaccine candidates in mice for protection against S. aureus infection but were not very successful [28,29]. A formalin-killed whole cell vaccine failed to protect animals in a rabbit endocarditis model. Whole cell vaccines made with irradiated wild-type and Spa mutant strains did not protect against subsequent intravenous infection with S. aureus. Recently, different whole cell preparations using mutant strains showed more promise in animal models. Immunization with an UV-irradiated preparation of a serine/threonine phosphatase mutant strain protected against systemic S. aureus infection [30]. Another study showed that previous immunization with a live strain containing a sortase A deletion protected against systemic infection [31]. In addition to intact whole cells, lysed cells have been shown to be protective in animal models [32–34]. It is important to note that these attempts were focused on antibody production against whole bacteria, and therefore T-cell (including Th17) responses, and their potential role in protection, were not evaluated. Here, we present evidence that a lysed S aureus whole cell vaccine preparation induces both antibody and Th17/Th1 responses when given with an adjuvant and can provide protection against S. aureus in three disease models. We show that while antiSaWCA antibody is sufficient for protection against skin dermonecrosis and invasive infection, passive transfer of antibodies did not protect against focal skin abscesses, arguing for an important and complimentary role of T-cells in protection following immunization with this vaccine.
2. Material and methods 2.1. Material Aluminum hydroxide (alum) was from Brenntag North America (2% Alhydrogel). Saline was from B. Braun Medical Inc. (Bethlehem,
PA). Cholera toxin (CT) was purchased from List Biological Laboratories. DMEM and FCS were from ThermoFisher scientific. Lysostaphin and other chemical reagents were purchased from Sigma. 2.2. Bacterial strains Staphylococcus aureus strains USA 300 TCH959 [35] and ATCC 29213 were purchased from ATCC. Bacteria were grown on blood agar plate overnight and then inoculated into Tryptic soy broth (TSB) to grow overnight at 37 °C with shaking. Cells were reinoculated into fresh TSB medium and incubated at 37 °C with shaking for 3 h. Cells were washed twice with saline and adjusted to concentrations as noted in animal models (described below) in saline before use. 2.3. SaWCA preparation The USA300 TCH959 strain was grown overnight on blood agar and resuspended into PBS. Cells were washed twice with PBS and resuspended to OD600 = 20 in PBS. Lysostaphin was added to the suspension and cells were shaken at 37 °C for 30 min. Cells were then lysed with sonication and then exposed to chloroform (1/40 vol/vol) and kept stirring at 4 °C for 2 h. Initially, we also included a preparation of cells that were only exposed to chloroform, without the initial lysing step. Either preparation was then plated on blood agar to confirm that no live bacteria was detectable before being frozen and then lyophilized. Lyophilized vials were reconstituted in the same volume of water and centrifuged for 5 min at 16,000g before use. For the lysed preparation, the supernatant fraction was isolated and defined as SaWCA. Protein concentrations were determined using the BCA kit with bovine serum albumin as standard (Thermo Scientific). 2.4. Immunization and challenge of mice All animal protocols were approved by the Institutional Animal Care and Use Committee at Boston Children’s Hospital (protocol number 16-03-3133). Female C57BL/6 J mice (Jackson Laboratories, Bar Harbor, Maine) were used for all experiments. The age at time of first immunization was between 4 and 6 weeks. Two types of adjuvants were used in mice immunizations. Cholera toxin (CT) was used as an adjuvant in intranasal immunizations to promote T cell responses to SaWCA whereas aluminium hydroxide (alum) was used for subcutaneous (s.c.) immunizations to promote both antibody and T cell responses [36–39]. For intranasal immunization, SaWCA was mixed with CT and the dose of immunization was 64 lg (protein content) of SaWCA and 1 lg of CT per mouse per immunization. Mice were immunized twice one week apart and bled three weeks after last immunization. Peripheral blood samples were stimulated with SaWCA and assayed for IL-17A production; plasma samples were analyzed for antibody production by ELISA. For s.c. immunization, vaccines were prepared as follows. One day prior to immunization, SaWCA was diluted to the appropriate concentration, and mixed with alum at the indicated concentration; the mixture was then rotated end-over-end overnight at 4 °C to allow for adsorption. The immunization dose in s.c. experiment was 100 lg of SaWCA (protein content) per mouse. Gently restrained, nonanesthetized mice received 3 s.c. injections of 200 ml of adjuvant with or without antigen in the back at 2-week intervals. Blood was drawn 2 weeks after the last immunization; plasma and whole blood were assayed for antibody and for IL17A and INF-c production as noted above. Mice were challenged in three different models as described previously with some modifications [40–42]:
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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a. Skin abscess model. To generate abscesses, the back of mice was shaved one day prior to infection, then anesthetized and injected s.c. with 2–5 105 CFU of strain USA 300 (vaccine strain). Abscesses become readily visible and palpable under the skin within 1–2 days. Mice were sacrificed on day 4 and abscesses were transferred into 0.5 ml of PBS and homogenized. Bacteria were plated on Mannitol salt plates and colonies were counted after overnight culture at 37 °C. b. Dermonecrosis model. In this model, the same procedure was used as for the skin abscess model, but with a higher inoculum of 5 106 1 107 CFU of freshly grown strain USA 300 (vaccine strain). Mice formed lesion by day 2 or day 3 and were monitored for 14 days and the surface area of the skin lesions was determined using pictures processed via Image J software. c. Intravenous sepsis model. Mice were anesthetized and infected with 2.5 107 CFU of strain ATCC 29213 via retroorbital injection. Mice were monitored twice daily for illness for 14 days. Any ill-appearing animal (including any signs of ruffled fur, slow moving or closed eyes) was immediately and humanely euthanized. For histopathology staining, skin tissues were removed and fixed in 10% neutral buffered formalin, and then paraffinembedded. Sections were stained with hematoxylin and eosin at
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the pathology core facility of Harvard Medical School. Pictures were taken using an Olympus microscope. These three different models were used to test the efficacy of the vaccine. In the abscess model, the infection is restricted to a local area and usually circumscribed inside an abscess (without any evidence of dissemination to blood or organs) as shown in Fig. 1A and C, a situation which mimics one of the most common manifestations of staphylococcal infections in humans. In the dermonecrosis model, the mice have more destructive lesions (Fig. 1B and D). The invasive disease model uses the encapsulated strain (ATCC 29213), which has also been used to evaluate the potential candidate antigen Als3p [19]. Thus the use of the three models allows for testing vaccine efficacy test in models with differing pathophysiology, strains and inoculum size. 2.5. Antibody production and passive immunization studies New Zealand white rabbits were immunized three times with 1 mg of SaWCA and Freund’s adjuvant at Cocalico Biologicals (Reamstown, PA). We chose rabbit serum as a source of antibody in the passive transfer experiments because it has been traditionally used in mouse passive experiment studies involving S. aureus and also because of the volume of sera required [43,44]. IgG antibody level against SaWCA remain above 50% of input for up to two weeks (data not shown), which exceeds the duration of these
Fig. 1. Histology of skin abscess and dermonecrosis lesions. Mice were infected with either 2 105 CFU of USA 300 (A and C) or 1 107 CFU of USA 300 (B and D) subcutaneously. Mice were sacrificed on day 4 and histological slides prepared for examination of the abscess (A) and dermonecrosis sites (B) as shown by arrows. Mice skins were fixed in 10% formalin and Hemotoxylin and Eosin-stained for abscess (C) and dermonecrosis (D).
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animal experiments. For passive transfer protection experiments, two different protocols were used. In the first, 500 ll of sera were given intraperitoneally 24 h before intravenous infection (sepsis model). To maximize any protective effect of antibodies, a second protocol was designed in which the bacterial inoculum was resuspended in 100 ll of sera for 30 min, after which the mixture of bacteria and sera was injected into mice (in all three models). 2.6. Enzyme-linked immunosorbent assay (ELISA) Assays for murine antibodies to whole bacteria were performed using Immulon 2 HB 96-microwell plates (Thermo Scientific, Waltham, MA) coated with SaWCA at 100 lg of protein per ml in PBS. Plates were blocked with 1% BSA in PBS. Antibody diluted in PBS with 0.05% Tween 20 (PBS-T) was added and incubated at room temperature for 2 h. Plates were washed with PBS-T, and secondary HRP-conjugated antibody to immunoglobulin G was added and incubated at room temperature for one hour. The plates were washed and developed with SureBlue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD). 2.7. Assay of IL-17A and IFN-c production in whole blood samples Fifty ml of heparinized blood were added to 450 ml DMEM (BioWhittaker, Walkersville, MD) containing 10% low-endotoxin defined FBS (Hyclone, Logan, UT), 50 mM 2-mercaptoethanol (Sigma) and ciprofloxacin (10 mg/ml, Cellgro, Manassas, VA). The cultures were incubated at 37 °C for 6 days with 10 lg/ml of SaWCA. Supernatants were collected following centrifugation and stored at -80 °C until analyzed by ELISA for IL-17A and INF-c (R&D Systems, Minneapolis, MN). For in vitro CD4+ and CD8+ depletion experiments, spleens and blood were obtained from immunized mice. Red blood cells were lysed with ACK lysis buffer (Thermo Fisher) and CD4+ or CD8+ cells were then depleted from the cell suspension with either anti-CD4+ or anti-CD8+ antibodylinked magnetic beads from Miltenyi Biotech following the manufacturer’s protocol. The remaining cells were plated at 1 106 cells/ml for PBMC and 1 107 cells/ml for splenocytes and stimulated with 10 lg/ml of SaWCA for 3 days before analysis for IL-17A production in the supernatant. 2.8. Opsonophagocytic killing assay (OPA) OPAs were performed as described previously [45] using differentiated HL-60 cells as a source of phagocytes. Briefly, USA 300 strain was grown as described above and resuspended to 2.5 104 CFU/ml in HBSS with 10% FBS. Eighty ll of bacteria (corresponding to about 2000 CFU) were first mixed with 10 ll of heatinactivated serum and incubated at room temperature for 30 min with shaking at 600 rpm. Ten ll of baby rabbit complement were added in the mixture. HL-60 cells that were differentiated using 0.8% N,N-dimethylformamide (DMF) were added at a cell:bacteria ratio of 100:1, shaken at 37 °C for another hour. Samples were diluted and plated on trypticase soy agar with 5% defibrinated sheep blood (TSA II) (BD) and incubated overnight at 37 °C with 5% CO2. Controls included bacteria treated in the absence of cells or with cells but no serum.
3. Results 3.1. Whole cell vaccine (SaWCA) preparations Killed non-lysed and lysed preparations were made as described above. In the case of the lysed preparation, the mixture was centrifuged at 16,000g for 5 min to remove intact cells and the supernatant fraction of killed whole cells was analyzed for protein composition on an SDS gel. The appearance of the supernatant fraction on the gel was virtually identical to that of the whole lysate (data not shown). Further analysis of the protein concentration by BCA showed that supernatant fraction retained about 80– 90% of the total protein amount (data not shown). 3.2. Lysed SaWCA is immunogenic following immunization by either the intranasal or subcutaneous route Our experience with S. pneumoniae suggested that intranasal immunization with killed cells adjuvanted with CT (an adjuvant known to promote Th17 responses [39,46]) could generate robust Th17 responses to protein antigens [47]. Therefore we began our experiments by testing whether the same would be observed with a killed staphylococcal whole cell vaccine. Surprisingly, as shown in the supplemental figure, although mice immunized with the killed non-lysed preparation intranasally twice a one-week interval generated higher Th17 response than CT-immunized mice, the level of IL-17A elicited by peripheral blood was quite low (median 25 pg/ml, see supplementary figure). In contrast, blood from mice that received the lysed preparation of SaWCA with CT expressed much higher IL-17A (median value 1640 pg/ml), also significantly greater than that of mice that received CT alone (median 8 pg/ml, P < 0.0001) (Fig. 2A). Given the reported importance of IL-17A in protection against staphylococcal infection [16,20], we selected the lysed preparation for all subsequent studies (which will be referred to as SaWCA). INF-c production by the blood of mice from the SaWCA/CT group after stimulation was significantly higher than that of mice that received CT alone, but overall low (median 22 pg/ml median 9.77 pg/ml, P = 0.015). The sera of SaWCA/CT-immunized mice had about 10 times higher anti-SaWCA antibody levels compared to the sera from mice immunized with CT (P < 0.001, Fig. 2B). Because we wished to elicit both robust antibody and T cell responses, subcutaneous immunization with alum as adjuvant was also evaluated, as we have previously shown that the addition of alum to pneumococcal whole cells enhanced cellular immune responses while also eliciting high antibody levels [48]. Mice subcutaneously immunized with SaWCA/alum generated significantly higher IL-17A (median 625 pg/ml) and IFN-c (median 48 pg/ml) responses than control mice (median 8 pg/ml and 8.54 pg/ml, P < 0.0001 and P < 0.05 respectively) (Fig. 2C). The antibody response to SaWCA was also significantly greater (by over 1000fold) in the SaWCA/alum-immunized compared to the control group, and 100 times higher than that observed following intranasal immunization with SaWCA and CT (Fig. 2D). The differential antibody response following intranasal vs. parenteral immunization with preserved T cell responses in both groups also provided an opportunity to evaluate the role of vaccine-elicited antibodies in each of the models.
2.9. Statistical analysis 3.3. IL-17A is generated by CD4+ cells Serum antibody concentrations, IL-17A and IFN-c release from peripheral cells and CFU counts in abscesses were compared between groups using the Mann-Whitney U test using PRISM (version 6.0 g, GraphPad Software, Inc). Differences in survival were analyzed by the Mantel-Cox test, using PRISM as well.
CD4+, CD8+, and cd T cells as well as innate lymphoid cells have all been shown to produce IL-17A [49–51]. We thus analyzed which cell type is the main source for IL-17A production in SaWCA-immunized mice. Blood PBMC or splenocytes from immu-
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production whereas depletion of CD8+ cells did not have any effect on IL-17A production. This result clearly suggests that IL-17A was mainly made from CD4+ T cells.
3.4. Binding and opsonophagocytic activity of sera obtained from mice immunized with SaWCA We then analyzed whether serum from SaWCA/alumimmunized mice generated antibody against staphylococcal antigens. We chose to evaluate the responses to 6 conserved antigens/virulence factors that had been previously shown to contribute to protection in various animal models: ClfA: clumping factor A; ClfB: clumping factor B; SdrD: SD-repeating containing protein D; a-toxin: alpha hemolysin; IsdA: Iron-regulated surface determinant protein A; IsdB: Iron-regulated surface determinant protein B [9,52,53]. As shown in Fig. 3A, SaWCA/alum immune serum demonstrated robust antibody responses to 3 proteins: SdrD, ClfA and ClfB and weaker, but still detectable, antibody responses to the other 3 proteins. Next we evaluated whether the sera from mice immunized with SaWCA/alum demonstrate in vitro killing activity. Two bacterial strains (USA 300 (vaccine strain) and ATCC 29213 (heterogenic strain)) were incubated first with heat-inactivated serum from
Fig. 2. Immune responses following intranasal and subcutaneous immunization with SaWCA. Blood samples from immunized mice were stimulated with 10 lg/ml (protein content) SaWCA for six days. IL-17A and INF-c were measured from cell supernatants for intranasal immunization (A) and subcutaneous immunization (C). Serum antibody against SaWCA was determined by ELISA following intranasal immunization (B) or subcutaneous immunization (D). E. Splenocytes or PBMC were depleted of CD4+ or CD8+ cells and stimulated with SaWCA for 3 days. Open bars represent cells without depletion, grey bars represent responses from cells lacking CD4+ cells and black bars represent responses from cells lacking CD8+ cells. Statistical analysis was done using the Mann-Whitney U test: *** P < 0.001, **** P < 0.0001.
nized mice were depleted with either CD4+ or CD8+ cells, and then stimulated with SaWCA. As shown in Fig. 2E, depletion of CD4+ cells totally abolished antigen-dependent stimulation of IL-17A
Fig. 3. Serological and functional analysis of immune responses following immunization with SaWCA and alum. (A) Antibody fold-rise in immunized mice against 6 selected surface proteins were determined by ELISA. (B) Opsonophagocytic killing assay using serum from mice immunized with SaWCA and alum vs. alum alone. Percentage of surviving bacteria from each sample is presented. Statistical analysis was performed using the Mann-Whitney U test: *** P < 0.001, **** P < 0.0001.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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are generated that not only can recognize important virulence factors of S. aureus, but also promote killing of S. aureus by neutrophils. 3.5. Protection by immunization with SaWCA in the three infection models
Fig. 4. Protection against skin abscess using SaWCA administered intranasally (A) or parenterally (B). The back of mice was shaved and mice subsequently challenged with 2 105 CFU of USA 300 for 4 days. Bacterial CFUs in abscesses were determined by serial dilutions. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Statistical analysis was done by MannWhitney U test: * P < 0.05, ** P < 0.01.
mice immunized either with alum or SaWCA/alum and then incubated with complement and HL-60-derived neutrophils. As shown in Fig. 3B, sera from mice immunized with SaWCA/alum killed 30% more bacteria than the control serum for both strains (P < 0.0001). Thus, following immunization with SaWCA and alum, antibodies
We first tested protection by SaWCA in the mouse abscess model. Immunization of mice with SaWCA/CT significantly reduced the bacterial density recovered from the abscess 4 days after infection compared to exposure to CT alone (Fig. 4A). Similarly, mice in the SaWCA/alum group also had a lower incidence of abscess compared to mice that received alum alone (10% vs. 70%, P = 0.02) and significantly fewer staphylococcal colonies could be recovered from the lesions when these were present (geometric mean 62 vs. 2.5 104 cfu, P = 0.0079) (Fig. 4B). In the dermonecrosis model, no protection was observed in mice immunized with SaWCA/CT compared to mice that received CT alone (Fig. 5A). In contrast, SaWCA/alum was clearly protective: the peak sizes (day 7) of the necrotic lesions were significantly lower in the SaWCA/alum-immunized mice compared to the controls (0.02 vs. 0.372 cm2, P = 0.0067), and similar differences were notable at most other measured time points (Fig. 5B). We have also examined skin pathology in the alum and SaWCA/alum immunized mice four days post infection. As shown in Fig. 5C, mice exposed to alum alone had disrupted skin tissue at the infection site and little infiltration of neutrophils can be observed. In contrast, SaWCA/ alum-immunized mice had intact skin tissue but an abscess that contains neutrophils and bacteria can be readily seen (Fig. 5D). We then evaluated whether protection against invasive disease following retroorbital injection of S. aureus would differ based on the immunization route. No protection was observed following
Fig. 5. Protection against dermonecrosis following immunization with SaWCA administered intranasally (A) or parenterally (B). Mice were infected with 1 107 CFU of USA 300 and the area of skin dermonecrosis was measured at different time points as indicated. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Skin histopathology with H&E staining was also performed on mice that received either alum (C) or SaWCA/alum (D) at four days post infection. Statistical analysis was calculated by Mann-Whitney U. * P < 0.05, ** P < 0.01, *** P < 0.001.
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intranasal immunization with SaWCA/CT (Fig. 6A) whereas SaWCA/alum-immunized mice had a significantly higher survival rate than mice immunized with alum alone (Fig. 6B, p = 0.045). Taken together, the result of these experiments suggest that antibodies, which are more robustly generated following parenteral immunization, play an important role in the dermonecrosis and sepsis models, but may be less critical in the abscess model. To evaluate this possibility further, we proceeded to evaluate whether protection could be achieved by passive immunization using sera directed against the lysate preparation.
3.6. Protection by passive transfer of immune serum To evaluate the role of antibodies in the different models used, we began our studies by injecting mice one day before infection with 500 ll of sera obtained from rabbits either before or after three immunizations with SaWCA. Although there was a trend towards delayed death (P = 0.07 by Kaplan-Meier) no difference in overall survival between the two groups of mice could be detected (2/10 vs. 0/10 survivors, Fig. 7A). To try to maximize protection provided by passive transfer, we pre-mixed bacteria with either pre- or post-immunization serum before infection. With this form of passive transfer, as shown in Fig. 7B, there was significant protection by immune serum (p = 0.03). The protective effect of immune serum clearly diminishes after 4 days, potentially due to the limited amount of antibody provided via this route (100 ll).
Fig. 6. Protection against sepsis following immunization with SaWCA administered intranasally (A) or parenterally (B). Mice were infected with 2.5 107 CFU ATCC 29213 intravenously and sickness was monitored for 14 days. The closed squares represent data from mice immunized with either CT or Alum; open squares represent data from mice immunized with SaWCA and adjuvant. Statistical analysis of the survival curves was performed by the Mantel-Cox test: * P < 0.05.
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Next we evaluated passive transfer of immune serum in the two other models. Immune serum did not protect mice in the skin abscess model with either infusion of 500 ll of immune serum one day prior to infection or bacteria pre-mixed with 100 ll of immune serum (Fig. 7C). When mice were challenged in the dermonecrosis model, mice that received immune serum had significantly better protection than that received pre-immunization serum after day 5, even though these mice had similar size of lesions early on (day 3 and 4) (Fig. 7D). Thus we conclude that antibodies directed against SaWCA contribute to protection in the sepsis and dermonecrosis model, whereas no effect of antibodies could be detected in the abscess model. 4. Discussion Given the morbidity and mortality associated with staphylococcal infections and the rise in antimicrobial resistance, there is an important need for the development of a vaccine for S. aureus. There is a growing consensus that a vaccine containing multiple components is in all likelihood required to combat this pathogen, given the complexity of the various toxins and other virulence factors produced by S. aureus [9,52–55]. Here, we present data using a lysed whole cell vaccine that is able to generate staphylococcusspecific antibody, Th17 and Th1 responses, and provide protection in three different mouse challenge models including a bacterial strain unrelated to the vaccine strain. Antibodies to key virulence factors were detectable in serum following immunization; the serum also promoted complement-mediated killing to two staphylococcal strains and was sufficient to confer protection against dermonecrosis and invasive disease. Whole cell vaccine preparations against S. aureus have been evaluated for protection in various preclinical models with marginal success, with more promising results when mutant strains (Spa or sortase mutants) were used [29–32]. A heat-killed whole cell vaccine was able to protect against intraperitoneal infection by live S. aureus and promoted phagocytosis [56]. A whole cell lysate vaccine containing 5 different strains of S. aureus designed to reduce dairy cattle mastitis has been commercially available for veterinary use in Europe [57,58]. Our strategy for whole cell preparation was also to lyse the bacteria to enhance immunogenicity, but in our case not by phage but by sonication. A key difference between our strategy and those employed previously is the introduction of adjuvants to promote both Th17 and Th1 responses. In the course of our work on the development of a whole cell or defined antigen pneumococcal vaccine, we found that when given with either alum hydroxide or CT, antigens elicited both antibody and Th17/Th1 responses, resulting in antibody-mediated protection against sepsis and IL-17A-mediated protection against nasopharyngeal colonization by S. pneumoniae [37,48,59,60]. Similarly, here, the inclusion of alum via the parenteral route induced Tcell and antibody responses whereas intranasal immunization with SaWCA and CT induced robust T-cell but only modest antibody responses. These results are consistent with those from our own group and that of others, in which intranasal immunization with CT elicits production of IL-17A and IFN- c but is less effective at generating systemic antibody responses than systemic immunization [36–39]. Alum-based adjuvants have being safely used in human for decades and avoid the potential safety issue of using intranasal cholera toxin mutants intranasally. Protection by SaWCA was tested against the original vaccine strain (USA 300) in both the abscess and dermonecrosis models and also against a heterogenic strain (29213), suggesting that protection is not restricted to the vaccine strain. However, protection against additional staphylococcal isolates needs to be tested to examine whether the protection by SaWCA is as broad as in the case of the pneumococcal whole cell vaccine [61].
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Fig. 7. Protection by passive transfer of sera. Mice were passively immunized with either pre-immune serum (closed squares) or post-immune serum (open squares) and then challenged in the sepsis model (A). Another two groups of mice were infected with strain 29213 resuspended in either pre-immune serum or post-immune serum (B). Similarly, mice were passively immunized with either pre-serum (closed squares) or post-immune serum (open squares) and then challenged in the abscess model (C), dermonecrosis model (D). Comparison of survival curves was performed using the Kaplan-Meier test; differences in bacterial CFUs and size of dermonecrotic lesions were analyzed using the Mann-Whitney U test.
Interestingly, the comparison of the two types of immunization suggests a differential role of antibodies and T cell responses in the two skin models we employed. Whereas both types of immunization resulted in protection against skin abscess, intranasal immunization with CT (which elicits comparable T cell responses, but significantly lower antibody responses than parenteral immunization with alum) did not provide significant protection against dermonecrosis, suggesting that antibodies may be most important in preventing this type of pathology. Along these lines, transfer of immune serum conferred protection in the dermonecrosis and invasive disease models, consistent with an important role of humoral immunity in preventing these infections. Conversely, the fact that passive transfer of immune serum did not protect mice in the abscess model supports the hypothesis that antibody is not critical in controlling this type of infection and that T-cell immunity may be the major factor for protection against skin abscesses. Another interesting implication of this study is that it may be possible to use the SaWCA as a probe for the identification of protective staphylococcal antigens, as we have done previously with S. pneumoniae [25,62]. The whole cell vaccine likely contains over 2000 staphylococcal proteins; thus, while quantification of each antigen in the whole cell vaccine and determination of their role in protection would represent a major challenge, more focused studies can be performed to examine a putative role of individual antigens of interest. For example, we were able to detect antibody
responses to a number of previously identified putative protective antigens: ClfA/B (fibrinogen-binding proteins that mediate attachment of S. aureus to host cells [63,64]), SdrD (a surface-associated calcium-binding protein against which antibodies have OPA) and Hla (alpha-hemolysin, an important virulence factor for skin dermonecrosis). Examination of preparations lacking these antigens could provide important information on their potential role in protection. We did not detect robust antibody responses against IsdA and IsdB, which suggests that an antibody response to these proteins may not be strictly necessary for protection against skin disease. Overall, we noted that while there was an over 1000 fold rise in antistaphylococcal antibody titer, the increase in killing capacity of the serum was only 30%, suggesting that only some of these antibodies contribute to OPA and that there are other antibodymediated mechanisms that provide protection. At the same time, it is conceivable that optimal protection against this organism may require eliciting both antibodies and T cell responses to a large number of antigens, which would be difficult to achieve with purified antigenic components. While killed whole cell vaccines are often considered an antiquated immunization approach, recent experience with S. pneumoniae suggests that the preclinical and clinical development of such a vaccine is feasible and promising [25,62,65]. Whole cell constructs also have the advantage of providing auto-adjuvanting effects which may enhance their immunogenicity and potency [59]. Thus, our
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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approach may have immunological advantages that should be further examined. Funding information This work was Pharmaceuticals.
supported
by
a
grant
from
Takeda
Acknowledgements YJL gratefully acknowledges support from Boston Children’s Hospital faculty development award and NIH grant R21AI103480 from the National Institute of Allergy and Infectious Diseases. RM gratefully acknowledges support from the Translational Research Program at Boston Children’s Hospital and NIH grant R01 AI100114. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine.2017.05. 085. References [1] Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus. J Clin Invest 2014;124:2836–40. [2] Zimlichman E, Henderson D, Tamir O, Franz C, Song P, Yamin CK, et al. Health care-associated infections: a meta-analysis of costs and financial impact on the US health care system. JAMA Int Med 2013;173:2039–46. [3] Magill SS, Edwards JR, Bamberg W, Beldavs ZG, Dumyati G, Kainer MA, et al. Multistate point-prevalence survey of health care-associated infections. N Engl J Med 2014;370:1198–208. [4] Wertheim HF, Melles DC, Vos MC, van Leeuwen W, van Belkum A, Verbrugh HA, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Inf Dis 2005;5:751–62. [5] Shinefield H, Black S, Fattom A, Horwith G, Rasgon S, Ordonez J, et al. Use of a Staphylococcus aureus conjugate vaccine in patients receiving hemodialysis. N Engl J Med. 2002;346:491–6. [6] Fattom A, Matalon A, Buerkert J, Taylor K, Damaso S, Boutriau D. Efficacy profile of a bivalent Staphylococcus aureus glycoconjugated vaccine in adults on hemodialysis: Phase III randomized study. Human vaccines & immunotherapeutics. 2015;11:632–41. [7] Rupp ME, Holley Jr HP, Lutz J, Dicpinigaitis PV, Woods CW, Levine DP, et al. Phase II, randomized, multicenter, double-blind, placebo-controlled trial of a polyclonal anti-Staphylococcus aureus capsular polysaccharide immune globulin in treatment of Staphylococcus aureus bacteremia. Antimicrob Agents Chemother 2007;51:4249–54. [8] Fowler VG, Allen KB, Moreira ED, Moustafa M, Isgro F, Boucher HW, et al. Effect of an investigational vaccine for preventing Staphylococcus aureus infections after cardiothoracic surgery: a randomized trial. JAMA 2013;309:1368–78. [9] Jansen KU, Girgenti DQ, Scully IL, Anderson AS. Vaccine review: Staphyloccocus aureus vaccines: problems and prospects. Vaccine. 2013;31:2723–30. [10] DeJonge M, Burchfield D, Bloom B, Duenas M, Walker W, Polak M, et al. Clinical trial of safety and efficacy of INH-A21 for the prevention of nosocomial staphylococcal bloodstream infection in premature infants. J Pediatr. 2007;151:260–5. 5 e1. [11] Weisman LE, Thackray HM, Steinhorn RH, Walsh WF, Lassiter HA, Dhanireddy R, et al. A randomized study of a monoclonal antibody (pagibaximab) to prevent staphylococcal sepsis. Pediatrics 2011;128:271–9. [12] Crum-Cianflone N, Weekes J, Bavaro M. Recurrent community-associated methicillin-resistant Staphylococcus aureus infections among HIV-infected persons: incidence and risk factors. AIDS Patient Care STDs 2009;23:499–502. [13] Ma CS, Chew GY, Simpson N, Priyadarshi A, Wong M, Grimbacher B, et al. Deficiency of Th17 cells in hyper IgE syndrome due to mutations in STAT3. J Exp Med 2008;205:1551–7. [14] Ishigame H, Kakuta S, Nagai T, Kadoki M, Nambu A, Komiyama Y, et al. Differential roles of interleukin-17A and -17F in host defense against mucoepithelial bacterial infection and allergic responses. Immunity 2009;30:108–19. [15] Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, et al. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest 2010;120:1762–73. [16] Archer NK, Harro JM, Shirtliff ME. Clearance of Staphylococcus aureus nasal carriage is T cell dependent and mediated through interleukin-17A expression and neutrophil influx. Infect Immun 2013;81:2070–5.
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[17] Joshi A, Pancari G, Cope L, Bowman EP, Cua D, Proctor RA, et al. Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model. Human Vaccines Immunother 2012;8:336–46. [18] Narita K, Hu DL, Mori F, Wakabayashi K, Iwakura Y, Nakane A. Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A. Infect Immun 2010;78:4234–42. [19] Spellberg B, Ibrahim AS, Yeaman MR, Lin L, Fu Y, Avanesian V, et al. The antifungal vaccine derived from the recombinant N terminus of Als3p protects mice against the bacterium Staphylococcus aureus. Infect Immun 2008;76:4574–80. [20] Montgomery CP, Daniels M, Zhao F, Alegre ML, Chong AS, Daum RS. Protective immunity against recurrent Staphylococcus aureus skin infection requires antibody and interleukin-17A. Infect Immun 2014;82:2125–34. [21] Choi SJ, Kim MH, Jeon J, Kim OY, Choi Y, Seo J, et al. Active immunization with extracellular vesicles derived from Staphylococcus aureus effectively protects against staphylococcal lung infections, mainly via Th1 cell-mediated immunity. PLoS One 2015;10:e0136021. [22] Brown AF, Murphy AG, Lalor SJ, Leech JM, O’Keeffe KM, Mac Aogain M, et al. Memory Th1 cells are protective in invasive Staphylococcus aureus infection. PLoS Pathog 2015;11:e1005226. [23] Glennie ND, Yeramilli VA, Beiting DP, Volk SW, Weaver CT, Scott P. Skinresident memory CD4+ T cells enhance protection against Leishmania major infection. J Exp Med 2015;212:1405–14. [24] Zhang Z, Clarke TB, Weiser JN. Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice. J Clin Invest 2009;119:1899–909. [25] Moffitt KL, Gierahn TM, Lu YJ, Gouveia P, Alderson M, Flechtner JB, et al. T(H) 17-based vaccine design for prevention of Streptococcus pneumoniae colonization. Cell Host Microbe 2011;9:158–65. [26] Malley R. Antibody and cell-mediated immunity to Streptococcus pneumoniae: implications for vaccine development. J Mol Med 2010;88:135–42. [27] Lu YJ, Gross J, Bogaert D, Finn A, Bagrade L, Zhang Q, et al. Interleukin-17A mediates acquired immunity to pneumococcal colonization. PLoS Pathog 2008;4:e1000159. [28] Greenberg DP, Ward JI, Bayer AS. Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits. Infect Immun 1987;55:3030–4. [29] van Diemen PM, Yamaguchi Y, Paterson GK, Rollier CS, Hill AV, Wyllie DH. Irradiated wild-type and Spa mutant Staphylococcus aureus induce anti-S. aureus immune responses in mice which do not protect against subsequent intravenous challenge. Pathogens Dis 2013;68:20–6. [30] Burnside K, Lembo A, Harrell MI, Klein JA, Lopez-Guisa J, Siegesmund AM, et al. Vaccination with a UV-irradiated genetically attenuated mutant of Staphylococcus aureus provides protection against subsequent systemic infection. J Infect Dis 2012;206:1734–44. [31] Kim HK, Kim HY, Schneewind O, Missiakas D. Identifying protective antigens of Staphylococcus aureus, a pathogen that suppresses host immune responses. FASEB J: Off Pub Fed Am Soc Exp Biol 2011;25:3605–12. [32] Camussone CM, Veaute CM, Pujato N, Morein B, Marcipar IS, Calvinho LF. Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant. Res Vet Sci 2014;96:86–94. [33] O’Brien CN, Guidry AJ, Douglass LW, Westhoff DC. Immunization with Staphylococcus aureus lysate incorporated into microspheres. J Dairy Sci 2001;84:1791–9. [34] Giese MJ, Adamu SA, Pitchekian-Halabi H, Ravindranath RM, Mondino BJ. The effect of Staphylococcus aureus phage lysate vaccine on a rabbit model of staphylococcal blepharitis, phlyctenulosis, and catarrhal infiltrates. Am J Ophthalmol 1996;122:245–54. [35] Highlander SK, Hulten KG, Qin X, Jiang H, Yerrapragada S, Mason Jr EO, et al. Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus. BMC Microbiol 2007;7:99. [36] Lu YJ, Zhang F, Sayeed S, Thompson CM, Szu S, Anderson PW, et al. A bivalent vaccine to protect against Streptococcus pneumoniae and Salmonella typhi. Vaccine 2012;30:3405–12. [37] Lu YJ, Yadav P, Clements JD, Forte S, Srivastava A, Thompson CM, et al. Options for inactivation, adjuvant, and route of topical administration of a killed, unencapsulated pneumococcal whole-cell vaccine. Clin Vaccine Immunol 2010;17:1005–12. [38] Lee JB, Jang JE, Song MK, Chang J. Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens. PLoS One 2009;4: e5190. [39] Mattsson J, Schon K, Ekman L, Fahlen-Yrlid L, Yrlid U, Lycke NY. Cholera toxin adjuvant promotes a balanced Th1/Th2/Th17 response independently of IL-12 and IL-17 by acting on Gsalpha in CD11b(+) DCs. Mucosal Immunol 2015;8:815–27. [40] Kim HK, Missiakas D, Schneewind O. Mouse models for infectious diseases caused by Staphylococcus aureus. J Immunol Methods 2014;410:88–99. [41] Molne L, Tarkowski A. An experimental model of cutaneous infection induced by superantigen-producing Staphylococcus aureus. J Invest Dermatol 2000;114:1120–5. [42] Krut O, Utermohlen O, Schlossherr X, Kronke M. Strain-specific association of cytotoxic activity and virulence of clinical Staphylococcus aureus isolates. Infect Immun 2003;71:2716–23.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085
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[43] Mishra RP, Mariotti P, Fiaschi L, Nosari S, Maccari S, Liberatori S, et al. Staphylococcus aureus FhuD2 is involved in the early phase of staphylococcal dissemination and generates protective immunity in mice. J Infect Dis 2012;206:1041–9. [44] McAdow M, Kim HK, Dedent AC, Hendrickx AP, Schneewind O, Missiakas DM. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood. PLoS Pathog 2011;7:e1002307. [45] Bagnoli F, Fontana MR, Soldaini E, Mishra RP, Fiaschi L, Cartocci E, et al. Vaccine composition formulated with a novel TLR7-dependent adjuvant induces high and broad protection against Staphylococcus aureus. Proc Natl Acad Sci USA 2015;112:3680–5. [46] Larena M, Holmgren J, Lebens M, Terrinoni M, Lundgren A. Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling. J Immunol 2015;194:3829–39. [47] Malley R, Lipsitch M, Stack A, Saladino R, Fleisher G, Pelton S, et al. Intranasal immunization with killed unencapsulated whole cells prevents colonization and invasive disease by capsulated pneumococci. Infect Immun 2001;69:4870–3. [48] Lu YJ, Leite L, Goncalves VM, Dias Wde O, Liberman C, Fratelli F, et al. GMPgrade pneumococcal whole-cell vaccine injected subcutaneously protects mice from nasopharyngeal colonization and fatal aspiration-sepsis. Vaccine 2010;28:7468–75. [49] Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004;21:467–76. [50] Xu H, Wang X, Liu DX, Moroney-Rasmussen T, Lackner AA, Veazey RS. IL-17producing innate lymphoid cells are restricted to mucosal tissues and are depleted in SIV-infected macaques. Mucosal Immunol 2012;5:658–69. [51] Gladiator A, Wangler N, Trautwein-Weidner K, LeibundGut-Landmann S. Cutting edge: IL-17-secreting innate lymphoid cells are essential for host defense against fungal infection. J Immunol 2013;190:521–5. [52] Spellberg B, Daum R. Development of a vaccine against Staphylococcus aureus. Semin Immunopathol 2012;34:335–48. [53] Proctor RA. Challenges for a universal Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:1179–86. [54] Proctor RA. Is there a future for a Staphylococcus aureus vaccine? Vaccine 2012;30:2921–7.
[55] Daum RS, Spellberg B. Progress toward a Staphylococcus aureus vaccine. Clin Infect Dis 2012;54:560–7. [56] Koenig MG, Melly MA, Rogers DE. Factors relating to the virulence of Staphylococci. III. Antibacterial versus antioxic immunity. J Exp Med 1962;116:601–10. [57] Luby CD, Middleton JR, Ma J, Rinehart CL, Bucklin S, Kohler C, et al. Characterization of the antibody isotype response in serum and milk of heifers vaccinated with a Staphylococcus aureus bacterin (Lysigin). J Dairy Res 2007;74:239–46. [58] Middleton JR, Ma J, Rinehart CL, Taylor VN, Luby CD, Steevens BJ. Efficacy of different Lysigin formulations in the prevention of Staphylococcus aureus intramammary infection in dairy heifers. J Dairy Res 2006;73:10–9. [59] Moffitt K, Skoberne M, Howard A, Gavrilescu LC, Gierahn T, Munzer S, et al. Toll-like receptor 2-dependent protection against pneumococcal carriage by immunization with lipidated pneumococcal proteins. Infect Immun 2014;82:2079–86. [60] Moffitt KL, Yadav P, Weinberger DM, Anderson PW, Malley R. Broad antibody and T cell reactivity induced by a pneumococcal whole-cell vaccine. Vaccine 2012;30:4316–22. [61] Malley R, Anderson PW. Serotype-independent pneumococcal experimental vaccines that induce cellular as well as humoral immunity. Proc Natl Acad Sci USA 2012;109:3623–7. [62] Moffitt KL, Malley R, Lu YJ. Identification of protective pneumococcal T(H)17 antigens from the soluble fraction of a killed whole cell vaccine. PLoS One 2012;7:e43445. [63] O’Brien L, Kerrigan SW, Kaw G, Hogan M, Penades J, Litt D, et al. Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB, the serine-aspartate repeat protein SdrE and protein A. Mol Microbiol 2002;44:1033–44. [64] McDevitt D, Francois P, Vaudaux P, Foster TJ. Identification of the ligandbinding domain of the surface-located fibrinogen receptor (clumping factor) of Staphylococcus aureus. Mol Microbiol 1995;16:895–907. [65] Moffitt KL, Malley R. Next generation pneumococcal vaccines. Curr Opin Immunol 2011;23:407–13.
Please cite this article in press as: Zhang F et al. Antibody-mediated protection against Staphylococcus aureus dermonecrosis and sepsis by a whole cell vaccine. Vaccine (2017), http://dx.doi.org/10.1016/j.vaccine.2017.05.085